Light & Fluorescence Microscopy
Simultaneous Imaging
Imaging more than one fluorescent probe at the same time (exciting with all required lasers and detecting the signal through all filters at the same time) is usually referred to as simultaneous image collection.
In a typical experiment simultaneously imaging the fluorescent probes DAPI and AlexaFluor 488 (AF488) involves exciting both fluorophores with both the 405nm and 488nm laser at the same time:
Simultaneous excitation of both DAPI and AF488.
The fluorescence signals resulting from dual excitation of these probes are usually collected in two separate detectors through two separate emission filters. The DAPI signal is collected through a 450/50nm bandpass filter. This signal should have very little signal emitted by the AF488 fluorophore (since the AF488 emission will not pass through the 450/50nm filter).
Emission spectra for DAPI and AF488. DAPI fluorescence is detected through a 450/50nm bandpass filter usually into the first detector
The AF488 signal is collected through a 525/50nm bandpass filter. However, as can be seen in figure_5, at least some of the fluorescence emission from both DAPI and AF488 will pass through the 525/50nm filter. In this example, almost 30% of the total signal detected in the second detector will originate from DAPI. Detection of more than one fluorescence signal in a single detector is usually referred to as spectral bleedthrough and it can be very difficult to separate the different fluorophore signals from each other.
Emission spectra for DAPI and AF488. DAPI fluorescence is detected through a 450/50nm bandpass filter (usually into the first detector) while AF488 fluorescence is detected through a 525/50nm bandpass filter (usually into the second detector). However, note that almost 30% of the signal seen in detector 2 originates from DAPI fluorescence.
Simultaneous imaging of more than one fluorescent probe has the advantages of more rapid image collection and, for live cell imaging, no temporal displacement between the two images. Good experimental design using adequately spectrally separated fluorophores can avoid (or at least minimise) spectral bleedthrough. An example of this would be imaging DAPI and AlexaFluor 647 (AF647)
Simultaneous imaging of DAPI and AF647 in a dual labelled specimen is possible without spectral bleedthrough because of minimal cross-excitation or emission of the two fluorophores.